RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1700179
D. R. Oliveira et al.
Archiv der Pharmazie
Table 2. Conditions to prepare compounds 1a and 1b.
Compound
Condition
Temperature (°C)
Time (h)
Yield (%)
1a
1b
Ultrasound
Conventional
25
90
0.5
3
97
60
Synthesis of compounds 3
Extracellular field recordings
To 2 mmol of compounds 2 in 5 mL of tetrahydrofuran, was
added 2 mL of aqueous NaOH 40%. The reaction was
maintained under vigorous stirring for 12 h at 90°C. The
solution was then neutralized with aqueous HCl, and
the solvent was removed under reduced pressure to dryness.
The crude solid was extracted with ethyl acetate, dried over
anhydrous Na2SO4, and the solvent evaporated again. The
product was purified by precipitation in ethyl acetate/hexane
mixture.
Field excitatory postsynaptic potentials (fEPSPs) were evoked
at 0.033 Hz using bipolar stimulating electrodes (0.05 mm
diameter) and recorded using glass micropipettes filled with
3 M NaCl (resistance 3–6 MΩ). Stimulating electrode was
placed in the Schaffer collateral and the recording electrode
was placed in the stratum radiatum (SR) of CA1 area. Once
responses were found, slices were fully submerged in
oxygenated aCSF perfused in a rate of 2 mL/min. A total of
50 mM of picrotoxin was also added to the perfusate in
all experiments. Stimulation intensity was selected that
produced responses that were around 50% maximal, and
experiments began after stable baseline response was
recorded for a period of at least 30 min. Responses were
measured and recorded online using WinLTP software
(version 2.20a; WinLTP Ltd. and The University of Bristol,
Bristol, UK). Compounds were applied by addition to perfuse
by 20 min.
3a: White solid (85%); 1H NMR (300 MHz, D2O, DSS, ppm) d
7.43–7.36 (m, 5H), 4.26–4.09 (m, 2H), 3.56–3.43 (m, 1H), 2.48 (d,
2H, J ¼ 6.4 Hz), 1.30 (d, 3H, J ¼ 6.9 Hz); 13C NMR (75 MHz, D2O,
DSS, ppm) d 176.1, 140.5, 128.1, 127.0, 126.9, 51.9, 47.6, 42.3,
19.5; HRMS calculated 193.1103; found 192.1205 [MꢃHþ].
3b: Yellowish liquid (80%); 1H NMR (300 MHz, D2O, DSS,
ppm) d 7.25–7.15 (m, 2H), 6.83–6.73 (m, 2H), 3.81–3.65 (m, 1H),
2.49 (dd, 1H, J ¼ 14.1, 5.1 Hz), 2.10 (dd, 1H, J ¼ 14.1, 8.4 Hz),
1.13 (d, 3H, J ¼ 6.4 Hz); 13C NMR (75 MHz, D2O, DSS, ppm)
d 176.1, 144.0, 128.9, 126.2, 119.3, 45.9, 42.3, 20.6; HRMS
calculated 213.0556; found 212.0658 [MꢃHþ].
Data analysis
Data were normalized to the mean slope recorded during the
30 min baseline. All data are presented as mean ꢁ standard
error of the mean (SEM) and EC50 are presented with 95%
confidence intervals in parentheses. Concentration–response
were plotted using GraphPad Prism software (version 6.0).
Standard agonist concentration–response curves were fitted
with the following equation: Y ¼ Ymin þ (Ymax ꢃ Ymin)/
(1 þ 10(logEC50-X)), where X is logarithm of the concentration
of the agonist and Y is the percentage inhibition. Ymin was
constrained to zero for curves.
Biological assays
Animals
Male Wistar rats 4 to 5-week-old were obtained from Charles
River, UK. All animals were housed under 12 h light/dark cycle,
and experiments were performed in the light phase of the
cycle. Animals were allowed access to food and water ad
libitum, and all procedures were carried out in accordance
with the Animals (Scientific Procedures) Act 1986.
Hippocampal slice preparation
The authors would like to thank Conselho Nacional de
ꢀ
ꢀ
Rats were killed by cervical dislocation and decapitation, and
the brains were rapidly removed and placed into ice-cold
artificial cerebrospinal fluid (aCSF; in mM: 124 NaCl, 3 KCl, 26
NaHCO3, 1.4 NaH2PO4, 1 MgSO4, 10 D-glucose, 2 CaCl2)
continuously oxygenated with 95% O2 and 5% CO2.
Parasagittal slices (400 mm) containing the hippocampal
formation were prepared in ice-cold, oxygenated aCSF using
Vibroslicer and placed in a Petri dish containing fresh-
oxygenated aCSF. The hippocampus was dissected from
these slices and area CA3 was removed to reduce the risk of
contaminating the field excitatory post-synaptic potential
(fEPSP) recording via indirect pathway to area CA1.
Hippocampal slices were left to rest for ꢄ2 h before
being transferred to the recording chamber maintained at
27–30°C, where they were continuously perfused with
oxygenated aCSF at a rate of 2 mL/min [22].
Desenvolvimento Cientıfico e Tecnologico, of the Ministry of
Science, Technology and Innovation of Brazil (CNPq, grant
~
455411/2014-0) and Sao Paulo State Research Foundation
(FAPESP, grant 2013/20479-9) for providing financial support
to JPSF’s lab. DRO also thanks CNPq for the post-doctoral
training grant (229217/2013-3).
The authors have declared no conflict of interest.
References
[1] D. L. Steer, R. A. Lew, P. Perlmutter, A. I. Smith,
M. I. Aguilar, Curr. Med. Chem. 2002, 9, 811–822.
[2] R. P. Cheng, S. H. Gellman, W. F. DeGrado, Chem. Rev.
2001, 101, 3219–3232.
ß 2017 Deutsche Pharmazeutische Gesellschaft
(6 of 7) e1700179